Bright‐field STEM tomography of thick biological sections

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By performing electron tomography in the scanning transmission electron microscope (STEM), it is possible to obtain 3D reconstructions at a resolution of around 10 nm from stained plastic‐embedded sections of eukaryotic cells in 1–2 µm thick sections. This is achievable because there are no imaging lenses after the specimen when the electron microscope is operated in STEM mode, so that chromatic aberration of the objective lens does not compromise the spatial resolution when there is strong multiple inelastic scattering [1‐4]. In STEM tomography it is necessary to minimize geometrical broadening of the probe by selecting a small probe convergence angle of ~1 mrad. Furthemore, by using an axial bright‐field detector instead of a standard high‐angle annular dark‐field detector, it is possible to reduce resolution loss caused by multiple elastic scattering in thick specimens [2‐4]. Recently, we have applied axial bright‐field STEM tomography to various biological problems, including: mechanism of vesicle release in presynaptic rod bipolar cell ribbon synapses in retina [5]; ultrastructural changes in postsynaptic densities in hypocampal neuronal cultures when specific scaffolding proteins are knocked out [6]; and ultrastructure changes that occur on activation of human blood platelets, small anucleate blood cells that aggregate to seal leaks at sites of vascular injury [7]. Electron tomograms were acquired with an FEI Tecnai TF30 transmission electron microscope equipped with a field‐emission gun and operating at an acceleration voltage of 300 kV. Specimens were prepared by conventional or freeze‐substitution techniques with osmium tetroxide fixation, and sections were cut to a thickness of between 1 µm and 1.5 µm and stained with uranyl acetate and/or lead citrate, before being coated with carbon and gold nanoparticles, which served as fiducial markers. Dual‐axis tilt series were acquired using a Gatan bright‐field STEM detector over an angular tilt range of ±60° and for some specimens ±68° with a 2° tilt increment. Tomograms were reconstructed using the IMOD program [8] and surface rendered using FEI Amira 3D software. The capabilities of bright‐field STEM tomography are illustrated in 3D reconstructions of blood platelets (Figure 1), which shows specimen and detector geometry, as well as orthoslices through a 1.5 µm thick section of a plastic‐embedded, frozen and freeze‐substituted preparation of platelets in early stage of activation. Of particular interest are the morphological changes that occur in alpha‐granules, which package important proteins that are released on platelet activation [9]. Structural changes that occur in the early stages of alpha‐granule activation are not understood due to difficulties in controlling the physiological state of platelets and in visualizing membranes at the nanoscale throughout entire platelets. Visualization of the 3D structure from the STEM tomogram (Figure 2) reveals that, on early activation, tubules extend from decondensing alpha‐granules and form pores with the plasma membrane, whereas other a‐granules remain in their condensed unactivated state [7]. These results from blood platelets, and a range of other biological systems, demonstrate that STEM tomography can visualize large cellular structures in their entirety at a spatial resolution of around 10 nm. In the case of blood platelets, it is possible to reconstruct complex interconnected membrane systems in almost complete cells.